Project Summary NK cells are capable of killing virus-infected and cancer cells and, therefore, play an important role in innate host defense. Unlike cytotoxic T lymphocytes (CTL), whose cytolytic activity is induced by productive TCR engagement with cognate pMHC ligands, activation of NK cells is regulated by a set of activating and inhibitory receptors. The net result of the signals induced by these receptors determines the extent of NK cell effector activities such as INF-? production and granule-mediated cytotoxicity. One of the main mechanisms of NK cell- mediated cytolytic activity depends on antibodies and is termed antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is induced by Fc?RIIIa (CD16) receptors that do not directly recognize antigens on the target cells, but interact with Fc fragments of IgG antibodies specifically bound to the antigens on the surface of target cells. Available evidence, including ours, strongly suggests that ligand-mediated clustering of activating receptors on cytotoxic lymphocytes regulates kinetics of intracellular signaling, formation of a highly ordered synaptic interface, kinetics of granule delivery, and efficiency of target cell lysis (1-6). The receptor clustering can be further modulated by antigen co-clustering with other membrane ligands on target cells, which interact with inhibitory receptors or adhesion molecules on the surface of NK cells. We hypothesize that differences in Fc?RIIIa (CD16) clustering/co-clustering could have significant impact on receptor-mediated signaling influencing efficiency of ADCC. Thus, the goal of this project is to study how homotypic and heterotypic ligand clustering regulates ability of the ligands to cooperate in the engagement of CD16, inhibitory receptors and adhesion molecules to modulate efficiency of ADCC. We will use well-characterized CD16.NK92 cells that we have developed to model functions of NK cells (7-12) and freshly activated human NK cells to investigate the role of CD16 clustering/co-clustering with other receptors. To achieve this goal, we will exploit fluorescent nanoparticles (NiNLPs) that can be loaded with ligands for CD16, adhesion molecules, and inhibitory receptors at various ratios and densities, and will examine the binding kinetics of these model membrane clusters to NK cells as well as the kinetics of induced intracellular Ca2+ signaling. We will also utilize planar lipid bilayers presenting the same ligands, either dispersed in the bilayers or co-localized by cross-linking with the iDimerize system, and will analyze how cross-linking of the ligands will influence structure of the synaptic interface and the kinetics of NK cell degranulation. The expected results will provide significantly improved understanding of the mechanisms controlling cytolytic activity of primary and activated human NK cells to design new NK cell-based immunotherapeutic strategies to improve efficiency of ADCC to treat viral infections, autoimmune diseases, and cancer.